Genome-wide identification and comparative analysis of the PYL gene family in eight Rosaceae species and expression analysis of seeds germination in pear

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of plant growth, seed germination, and stress responses. The pyrabactin resistance 1-like (PYR/PYL) protein, an ABA receptor, was involved in the initial step in ABA signal transduction. However, the evolutionary history and characteristics of PYL genes expression remain unclear in Chinese white pear (Pyrus bretschneideri) or other Rosaceae species. In this study, 67 PYL genes were identified in eight Rosaceae species, and have been classified into three subgroups based on specific motifs and phylogenetic analysis. Intriguingly, we observed that whole-genome duplication (WGD) and dispersed duplication (DSD) have a major contribution to PYL family expansion. Purifying selection was the major force in PYL genes evolution. Expression analysis finds that PYL genes may function in multiple pear tissues. qRT-PCR validation of 11 PbrPYL genes indicates their roles in seed germination and abiotic stress responses. Our study provides a basis for further elucidation of the function of PYL genes and analysis of their expansion, evolution and expression patterns, which helps to understand the molecular mechanism of pear response to seed germination and seedling abiotic stress. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08456-1.


Introduction
Abscisic acid (ABA) plays a pivotal role in various aspects of plant growth and development, such as cell elongation and division, seed desiccation tolerance and dormancy, seed maturation and germination, root growth, leaf senescence, fruit ripening and adaptation to different biotic and abiotic stresses [1][2][3][4]. Moreover, ABA can crosstalk and interact with other phytohormones to regulate plant development and response to environmental cues [5,6].
In plants, the ABA receptors Pyrabactin Resistance 1 (PYR1), PYR1-Like (PYL), and Regulatory Component of ABA Receptor (RCAR) (hereafter referred to as PYLs) function at the first step of the ABA signal pathway and activate downstream ABA signaling cascade [7,8]. After binding ABA, PYLs subsequently interact with the clade A of protein phosphatase type 2Cs (PP2Cs). In the absence of ABA, PYL protein cannot bind to PP2C, thus preventing the activation of SUCROSE NONFERMENTING 1 (Snf1) -related protein kinase 2s (SnRK2s); whereas upon binding ABA, PYL can interact with PP2C and inhibits PP2C from dephosphorylating SnRK2 [9,10]. In turn, phosphorylated SnRK2 activates downstream substrates,

Identification, phylogenetic relationship and features of PYL genes
To identify all the PYL gene members in eight Rosaceae species (Chinese white pear, European pear, Japanese apricot, apple, peach, strawberry, sweet cherry and black raspberry), the HMM (Hidden Markov Model) and BLASTp (e-value < = 1e-10) searches were carried out to search genome annotation using 14 Arabidopsis thaliana PYL [18] amino acid sequence as queries. The candidate PYL genes were validated by Pfam (http:// pfam. xfam. org/) and Interproscan 63.0 (http:// www. ebi. ac. uk/ Inter ProSc an/). The methods of screening and identification were referred to previous reports [19,30]. After filtering out redundant and incomplete protein sequences, a total of 67 PYL genes were determined: 11 in Chinese white pear, 7 in European pear, 7 in Japanese apricot, 13 in apple, 7 in peach, 8 in strawberry, 6 in sweet cherry and 8 in black raspberry (Supplementary Table 1).
To understand the phylogenetic relationship of the PYL proteins, the amino acid sequences encoded by PYL genes from eight Rosaceae species and Arabidopsis were used to construct a phylogenetic tree with the Neighbor-Joining (NJ) algorithm. Based on the classification of subfamilies in Arabidopsis [18], all the PYL genes in eight Rosaceae species were classified into three clades, designated Subfamily I, Subfamily II and Subfamily III (Fig. 1A). Overall, Subfamily I contained 22 members, Subfamily II contained 23 PYLs, and Subfamily III comprised 36 PYLs (Fig. 1A). PYLs genes grouping into the same subfamilies may perform similar functions. The PYL genes of Chinese white pear were named at PbrPYL1-11 according to clustering relationship with Arabidopsis 14 AtPYL genes (AtPYR1 and AtPYL1- 13) in order. PbrPYL2-11 were randomly distributed on 8 of 17 chromosomes and PbrPYL1 on scaffold984.0 ( Fig. 1A and Supplementary Table 1).
The MEME analysis was used to verify the classification of phylogenetic tree by analyzing the presence of conserved motifs in 67 PYL family members. 13 conserved motifs were detected from 67 PYL proteins, all of which contained motif 1, motif 2 and motif 3 (Fig. 1B). Remarkably, each of three subfamilies contained similar motifs; motif 4 was specific to subfamily I and motif 7 was only appeared in subfamily III, indicating that each subfamily is characterized by their respective conserved motifs, which may contribute to unique/specialized biological functions. All the PbrPYL genes include the PYR_PYL_RCAR domain ( Supplementary Fig. S1). All  II consists of two introns,  whereas no introns were detected subfamilies I and III except PbrPYL4 (Supplementary Fig. S1). Different PYL genes were grouped into each subfamily depicting similar motifs, domains and exon-intron structures, which confirmed their close evolutionary relationships and the classification of subfamilies. The detailed characteristics of the PYL proteins of Arabidopsis and eight Rosaceae species were listed in Supplementary  Table 1).
In addition, to better understand the potential function of the PbrPYL genes, we extracted promoter sequences of 2000 bp upstream from the initiation codons and identified cis-elements using the PlantCARE database. Some common cis-regulatory elements were briefly marked in promoter region, and the diversities of cis-elements related to the light, plant hormone and regulatory stress response suggested that expression may differ in response to development and stress ( Supplementary Fig. S1).

Synteny analysis of PYL gene family
Several gene duplication modes contribute to the formation of local gene clusters and evolution of protein-coding gene family, such as WGD, tandem and segmental [31]. Different modes of gene duplication were identified in eight Rosaceae species, and we detect duplicated PYL gene pairs to infer the evolutionary origins. All the PYL family members were assigned to five modes of gene duplication, including segmental duplication/ WGD, tandem duplication, proximal duplication, transposed duplication and dispersed duplication (Table 1 and Supplementary Table 2). A total of 87 duplicated gene pairs of PYL family members were identified in eight Rosaceae species, and the largest number is dispersed duplication (57 gene pairs, 65.5%), followed by WGD (26 gene pairs, 29.9%), and other duplications (4 gene pairs, 4.6%), suggesting that the modes of dispersed duplication and WGD mainly contribute to the expansion of the PYL gene family. Remarkably, pear and apple experienced the recent WGD event [32][33][34], so the number of PYL genes was more abundant in Chinese white pear and apple than in other species. The number of WGD duplications in apple and Chinese white pear were 11 and 6, but there were just one in Japanese apricot, European pear, black raspberry and strawberry. The number of dispersed duplication accounted for a large proportion of replication modes in each Rosaceae species, with the highest percentage in Japanese apricot and European pear (85.7%), and with the lowest percentage in apple (47.6%). Both WGD and dispersed duplication events play important roles in apple and Chinese white pear. The dispersed duplication events occurred more frequent than WGD in Rosaceae species except apple.
The PYL family genes were randomly distributed on different chromosomes in each species. For Chinese white pear, 10 of 11 PbrPYL genes were distributed on 8 of the 17 chromosomes while PbrPYL1 was located on scaffold984.0 ( Fig. 2A and Supplementary Table 1). Furthermore, intra-genomic collinearity of PYL gene family was investigated in each of eight Rosaceae species. 6 pairs was found in Chinese white pear, 3 pairs in European pear, 11 pairs in apple, 2 pairs in Japanese apricot, and one pair in strawberry, peach, sweet cherry and black raspberry ( Fig. 2A-H and Supplementary Table 2). Further, the collinear correlation of the PYL genes between Chinese white pear and the other seven Rosaceae species were identified. A total of 81 pairs were found including 13, 1, 13, 13, 14, 12 and 14 collinear gene pairs between Chinese white pear with strawberry, apple, sweet cherry, Japanese apricot, peach, European pear and black raspberry ( Fig. 2I and Supplementary Table 2). The results suggest that a conserved collinearity relationship between Chinese white pear and the other seven Rosaceae species.

PYL genes evolved under strong purifying selection
Deleterious mutations can be eliminated by negative (purifying) selection and advantageous mutations can be accumulated by positive (Darwinian) selection [35]. To detect the selection pressure of PYL genes, we estimated the Ka, Ks and Ka/Ks values of paralogous PYL gene pairs in the eight Rosaceae species ( Table 2). The strength and direction of selection pressure have been widely measured based on Ka/Ks ratio (Ka/Ks > 1: positive selection; Ka/Ks = 1: neutral evolution; Ka/Ks < 1: negative selection) [36]. All Ka/Ks ratios of the paralogous genes were less than one ( Table 2), suggesting that purifying selection was the main driving force of PYL family gene evolution in Rosaceae species.

Expression pattern of PYL family genes in different pear tissues
To analyze the expression levels of PYL family genes in different pear tissues, previously published RNA-seq

Expression analysis of PbrPYLs during ABA treatment and seed germination
Previous research has shown that dormant seed coats produce ABA to suppress embryo germination [37,38]. Therefore, pear seed coats were peeled to prevent ABA interference and accelerate germination. As expected, the uncoated seeds treated with water successfully germinated in 36 h (Fig. 4A). However, the seeds treated with ABA for 36 h did not germinate as well as control treated for 0 h (Fig. 4A). As the ABA receptors, the PYL family has been reported to play an important role in seed germination [18,39]. To better reveal the functions of PYLs in the ABA signal pathway during the seed germination, 11 PbrPYL genes were detected using qRT-PCR. Consistent with the transcriptome data in seed (Fig. 3), just PbrPYL7, Pbr-PYL8 and PbrPYL9 genes of the 11 PYL genes were much greater expressed than those of the other PYLs expressed in seeds (Fig. 4B). Moreover, PbrPYL7, PbrPYL8 and Pbr-PYL9 of subfamily II were significantly lower expressed in the germinated embryos (36 h-H 2 O) than in the dormant embryos (0 and 36 h-ABA treatment) (Fig. 4B). Conversely, the expression of other PbrPYL genes except the PbrPYL7, PbrPYL8 and PbrPYL9 genes were low or and almost not expressed. Although the low expression of Pbr-PYL2, PbrPYL3, PbrPYL4, PbrPYL5, PbrPYL6, PbrPYL10 and PbrPYL11, they were most strongly expressed in the germinated embryos than dormant embryos (Fig. 4B). The expression of PbrPYL2, PbrPYL3, PbrPYL4, PbrPYL5, PbrPYL6, PbrPYL10 and PbrPYL11 increased dramatically at 36 h after germination, whereas that of PbrPYL1, PbrPYL7, PbrPYL8 and PbrPYL9 decreased substantially. Upon exogenous ABA treatment during germination, the trends of gene expression were somewhat changed (Fig. 4B). Therefore, ABA can inhibit germination of the uncoated seed. Meanwhile, this significant change of Pbr-PYL genes indicates that PYLs play critical and various roles in ABA-mediated seed germination.

Expression analysis of PbrPYLs in pear seedling under different treatments
In this study, the expression patterns of 11 PbrPYL family genes were detected by qRT-PCR, and the results showed transcriptional changed under heat, cold, drought, NaCl,   (Fig. 5). Under cold treatment, the genes of PbrPYL1, PbrPYL2, PbrPYL7, PbrPYL8, PbrPYL9, PbrPYL10 and PbrPYL11 did not change significantly at 6 h after treatment, but up-regulated at 24 h (Fig. 5). Under drought or NaCl treatment, most genes had similar expression patterns, PbrPYL7, PbrPYL8 and Pbr-PYL9 genes were significantly up-regulated at 12 h, while there was no significant difference at 24 h. Other genes were significantly up-regulated at 24 h (Fig. 5). Under ABA treatment, the genes of PbrPYL1, PbrPYL2, PbrPYL5, PbrPYL7, PbrPYL10 and PbrPYL11 were upregulated rapidly at 6 h. All the genes except PbrPYL5 were significantly up-regulated at 24 h (Fig. 5). In addition, three highly expressed genes, PbrPYL7, PbrPYL8 and PbrPYL9, had similar expression trend at 12 h under heat, drought and NaCl treatments, and observably upregulated at 24 h under cold and ABA treatments. Taken together, all the nine genes were induced by different stresses, but the diverse expression pattern of PbrPYL genes may suggest that these genes may be critical to abiotic and hormone stress responses.

Subcellular localization of PbrPYL proteins
To investigate the subcellular localization of the PbrPYL proteins, three highly expressed genes PbrPYL7/8/9 were cloned and individually constructed into a recombinant plasmid. Microscopic visualization showed that the GFP fluorescence signal of the positive control 35 S-GFP was distributed the whole cell, whereas the fluorescence of PbrPYL7/8/9-GFP was detected in the cytosol and the nucleus (Fig. 6). The results showed that PbrPYL proteins were localized in both the cytoplasm and the nucleus, which is consistent with the previous research [20,27]. PP2C protein is at the downstream of PYL protein and interacts with PYL, which is located in the nucleus [27]. PP2C complexes were also localized to the nucleus, even if the interacting partners PYL protein were not exclusively localized in the nucleus [20,27]. This indicates that the PYL may function as a nuclear ABA receptor in the regulation ABA-dependent gene expression.

Gene structure and evolution
ABA is directly perceived by the PYL receptor, which plays an important role in initiating the ABA signaling pathway [18,40]. The identification and function of PYL genes have been studied in many model species, such as Arabidopsis [7], Oryza sativa [15,27] and Solanum lycopersicum [41]. However, the knowledge of the PYL gene family is very limited in the Rosaceae species. In present study, a genome wide comprehensive analysis of PYL genes from 8 Rosaceae species was carried out, and their potential role in pear seed development was subsequently investigated. A total of 67 PYLs in the 8 Rosaceae species were identified based on the similarity to 14 AtPYLs. The numbers of PYLs across 8 different Rosaceae species ranges from 6 to 13, including 13 in apple, 11 in Chinese white pear, 8 in strawberry, 8 in black raspberry, 7 in European pear, 7 in Japanese apricot, 7 in peach and 6 in sweet cherry. Meanwhile, chromosomal distribution of PYL genes across 8 Rosaceae species showed uneven distribution (Fig. 2). Based on phylogenetic analysis with Arabidopsis homologs, 67 PYLs can be broadly classified into three clusters: subfamilies I, II and III (Fig. 1A), which is consistent with the classification of PYL genes in other plant species [28,29]. The number and composition of conserved motifs varied in each PYL subfamilies (Fig. 1B). All 67 PYL protein contained motif 1, motif 2 and motif 3, indicating a highly conserved function of this family. Phylogeny analysis of PYL genes showed that similar motifs exist in each subfamily, such as motif 4 and motif 7 were specific to subfamily I and subfamily III, respectively (Fig. 1). PYL genes of the same subfamily have similar unique/specialized biological functions, but their functions remain unclear. Gene duplication events can be generally divided into five types, including WGD, TD, PD, DSD and TRD, which could drive gene family expansion in eukaryotes  [42,43]. WGD events can generate a large number of duplicate genes in a short period of time [44]. WGD and small-scale duplication events are not only the main features of eukaryotic genome evolution, but also the main driving force of new functions in the genome and genetic evolutionary system [43]. Gene families, such as heatshock transcription factors, SWEET and F-box families, expanded primarily through WGD and DSD [45][46][47]. However, the expansions of WRKY and AP2/ERF gene families mainly resulted from TD events [48,49]. In this study, WGD and DSD were found to be the main driving forces for the expansion of the PYL gene family in 8 Rosaceae species. Meanwhile, the number of DSDs account for a large proportion of gene duplications and made primary contributions in the expansion of PYL genes (Table 1). In addition, evolutionary analysis based on the Ka, Ks and Ka/Ks, suggesting that purifying selection were the primary evolutionary force imposing on PYL family genes in 8 Rosaceae species ( Table 2).

Regulation of gene expression
Analyzing the expression pattern of pear PYL genes across the broad spectrum of various tissues, developmental stages and stress treatments will help further understand the physiological and developmental functions of PbrPYL. The PYL genes expression pattern in different tissues has been studied in many species. The expression levels of most PYL genes in soybean seeds were higher than those in other tissues [50]. The transcription level of PYLs showed much higher in seeds of oilseeds during germination than in other various tissues [51]. The transcription level of PYLs was highly abundant in latex of rubber tree [52]. PYLs were expressed at a higher level in the callus of B. rapa than in other tissues [53]. We analyzed the PbrPYL genes expression patterns in the various tissues of 'Dangshang' pear. The result shows that PbrPYL1, PbrPYL4, PbrPYL5, PbrPYL6, PbrPYL7, PbrPYL8 and PbrPYL9 showed higher expression potential in different tissues, suggesting that these PYLs have multiple roles throughout the growth and development. Furthermore, we found that all 3 members (PbrPYL7, PbrPYL8 and PbrPYL9) of subfamily II showed much higher expression potential in different tissues (Figs. 3 and 4). However, other PbrPYL genes in all of the various tissues were nearly zero, indicating that the functions of these PbrPYL genes are not required (Fig. 3). Meanwhile, the results showed that only PbrPYL1, PbrPYL4, PbrPYL7, PbrPYL8 and PbrPYL9 were highly expressed in dormant embryos (Fig. 3).
ABA acts as a primary mediator in seed dormancy and germination [37,38]. In this study, uncoated seeds germinated for 36 h under water treatment, but there was no significant change at the same time under ABA treatment (Fig. 4A). This indicates that ABA inhibited seeds germination, which was consistent with previous studies [24,38]. PYL gene family as ABA receptors starts from ABA sensing and plays an important role in ABA-mediated seed germination. The OsPYL7, OsPYL8 and OsPYL9 in rice have been shown to play important role during seed germination and development stage [19]. OsPYL/RCAR5 and PYL8/RCAR3 may perform critical biological functions in seed germination and seedling growth in rice and Arabidopsis [27,54]. PYL11 and PYL12 were expressed specifically in mature seeds, which positively modulate ABA-mediated seed germination in Arabidopsis [8]. The Arabidopsis pyr1, pyl1, pyl2 and pyl4 single mutants were not sensitive to ABA during seed germination, while they quadruple mutant was strongly insensitive to ABA [18]. PYL13 overexpression mutants were sensitive to ABA during seed germination [39]. PYR1, PYL1, PYL2 or PYL4 overexpression mutants can increase sensitivity to ABA-mediated inhibition during seed germination [55]. Among the 11 PYL genes detected in pear, the expression of 7 PbrPYL were up-regulated during seed germination and 4 PbrPYL were down-regulated by exogenous ABA. This result indicates the diversity of the expression patterns and multiple roles of PYLs in ABA signaling.
ABA has been reported to play crucial roles in responding to multiple abiotic stresses, such as heat, cold, drought and salinity [56,57]. ABA was produced rapidly in response to multiple stresses and then regulates the stress response. PYL as the ABA receptor was involved in the initial step under multiple abiotic stresses [20,28,29]. In this study, qRT-PCR analysis showed that all the PbrPYLs were almost up-regulated by one or more different abiotic stresses (heat, cold, drought and NaCl) and ABA treatment at seedling stage, except PbrPYL4 and PbrPYL6, the expression of which were too low to be detected. This suggests that PYLs play important roles in stress responses and ABA treatment of pear. By contrast, the expression patterns of all 3 subfamily II members (PbrPYL7, PbrPYL8 and PbrPYL9) were up-regulated many folds than control under four different abiotic stresses and ABA treatment. These PbrPYLs may be utilized to improve the tolerance of pear seedling under abiotic stresses and ABA treatment.

Conclusions
In conclusion, 67 PYL genes were identified in eight Rosaceae species and classified into three subgroups. WGD and DSD were major contributions to PYL family expansion. Purifying selection was the major force in PYL gene evolution. The qRT-PCR analyses of 11 PbrPYL genes revealed multifaceted critical roles of PYL in seed germination as well as abiotic stress responses. This study provides a basis for further elucidation of the function of PYL genes and analysis of their expansion, evolution and expression patterns, which helps to understand the molecular mechanism of pear in response to seed germination and seedling abiotic stress.

Identification of PYL genes in pear and other Rosaceae species
To identify the PYL gene family in Chinese white pear and other seven Rosaceae species, multiple database searches were performed. The Chinese white pear (Pyrus bretschneideri) genome sequence was retrieved from the Pear Genome Project (http:// pearg enome. njau. edu. cn/) [

Phylogenic and conservative motif analysis of PYL family members
The phylogenetic trees were constructed by MEGA7.0 [58] using the full-length protein sequences of PYL from eight Rosaceae species. Neighbor-Joining (NJ) algorithm with a matrix of pairwise distances estimated was performed for amino acid sequences. 1000 replicates were carried out for Bootstrap analysis. All full-length amino acid sequences of the PYLs were analyzed for conserved motifs by online MEME (http:// meme-suite. org/). The conserved domains of PYL were analyzed by online Conserved Domain Database (CDD) (https:// www. ncbi. nlm. nih. gov/ cdd). The gene structure of PbrPYLs was analyzed online by the GENE Structure display (GSDS 2.0) website (http:// gsds. gao-lab. org/). The 2000 bp genomic sequences upstream of PbrYPLs were extracted, and the cis-acting elements in the promoter region were analyzed by PlantCARE (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/).

Chromosomal location and synteny analysis of PYL genes
Chromosomal location information of PYL genes were obtained from genome annotation files of eight Rosaceae species and displayed using the TB-tools software [59]. The synteny analysis among eight Rosaceae genomes was performed using the method implemented in the PGDD (http:// chibba. agtec. uga. edu/ dupli cation/) [60]. Potential homologous gene pairs were identified using BLASTP (E < 1 e-5, top 5 matches). And the homolog pairs and gene location information were analyzed by MCScanX to identify syntenic chains [61]. Afterward, DupGen_finder was further employed for whole-genome (WGD), tandem (TD), proximal (PD), transposed (TRD) and dispersed (DSD) duplications of PYL family genes [62]. The results were visualized using the TB-tools software [59].

Calculating values of Ka, Ks and Ka/Ks
The nonsynonymous (Ka) and synonymous (Ks) substitution rates and Ka/Ks ratios were calculated for valid gene pairs by KaKs_Calculator 2.0 with a model-averaged method and default parameters [63].

Expression analysis of PYL in different tissues
The transcriptome data of 'Dangshansuli' different tissues were used to analyze the expression patterns of PYL family members. The transcriptome data of different tissues were obtained from our previously published studies and unpublished data [34,64,65], including pollen, seed, petal, sepal, ovary, stem, bud, leaf and fruit. RNA-Seq raw data were obtained from the Sequence Read Archive (https:// www. ncbi. nlm. nih. gov/ biopr oject/) with the accession numbers PRJNA503323 and PRJNA498777 [64]. The RPKM values were used to estimate the gene expression abundances.

Plant materials treatment and qRT-PCR analysis
'Cuiguan' pear (Pyrus pyrifolia Nakai) seeds were obtained from the pear germplasm orchard of Pear Engineering Technology Research Center of Nanjing Agricultural University situated at Baima in Nanjing with the permission. Seeds with seed coat peeled were treated with water or 1 ppm ABA. Seeds were placed on moistened gauze in a growth chamber at 25 ± 1 °C with dark and 60% relative humidity, and then collected in 0 h, 36 h (ABA treatment) and 36 h (water treatment). Germinated seeds were sowed in plastic pots. Seedlings were grown in a growth chamber for five weeks (the photoperiod 16/8 h, the temperature 25 ± 1 °C) and then exposed to various stresses. Seedlings were exposed to 4 and 37 °C for cold and heat treatment, respectively. For NaCl, drought stress and ABA treatments, the seedlings were cultured in 200 mM NaCl, 20% PEG 6000, and 100 µM ABA. The conditions of stress treatments were referenced by previous reports [19]. All treated samples and blank controls were collected in continuous time intervals of 6, 12 and 24 h, respectively. The samples were immediately frozen in liquid nitrogen, and stored at − 80 °C until use. Total RNA was extracted using RNAprep Pure Plant Kit (Tiangen, Beijing, China). The extracted total RNAs were synthesized the first-strand cDNA using TransScript One-Step gDNA Removal and cDNA synthesis Supermix (TransGen, Beijing, China). The primers of all the PYL family genes were designed using Primer Premier 6.0 and listed in Supplementary Table 4. The qRT-PCR was carried out using a LightCycler 480 SYBRGREEN I Master (Roche, USA). The tubulin gene of pear was used as the reference. All reactions were carried out with three independent biological replicates. The genes expression levels were calculated with the 2 −ΔΔCt method.

Subcellular localization of the PYL genes
The full-length PbrPYL coding sequences without the termination codon were cloned from pear, and directionally inserted into the pCAMBIA1300-35 S: CDS-GFP vector. Primers used for cloning were listed in Supplementary Table 4. The recombinant plasmids and the control plasmid (pCAMBIA1300-35 S alone) were individually transformed into Agrobacterium tumefaciens strain GV3101. 30-day-old tobacco (Nicotiana benthamiana) leaves were used for agrobacterial injection according to the published protocol [66]. DAPI staining was used to indicate the nucleus. The fluorescence was imaged using a confocal microscope LSM780 (Zeiss LSM 780, Germany).